1. Technical Field
The present invention is related to soluble interleukin-2 receptor (IL-2R) and a method of assaying the same. More particularly, the present invention is related to the discovery of soluble, IL-2R in human body fluid as a result of disturbed body function and a solid phase immunoaassay for quantitative determination thereof.
2. State of the Art
The ligand interleukin-2 (IL-2) and its receptor (IL-2R) per se, are well known entities. Interleukin-2 or T-cell growth factor as it is sometimes called, is a lymphokine, synthesized and secreted by lectin--or antigen-activated T-cells. The reported biological activities of IL-2 include stimulation of long-term in vitro growth of activated T-cell clones, induction of cytotoxic T-cell reactivity and the like. It is believed that IL-2 exerts its biological effects by interacting with specific high affinity receptors (IL-2R) on the surface of activated T-cells.
In contrast to hormone mediated systems, cellular activation is a prerequisite for the induction of both the ligand (IL-2) and its receptor (IL-2R), the latter being expressed rapidly at the cell surface in a time-dependent and heterogeneous manner (Cantrell, et al, Science, 224: 1312-1316, 1984).
When certain human cells are stimulated or otherwise activated, they manufacture and express newly synthesized molecules on their cell surfaces and also release these molecules in a soluble form. Hence, the detection of these molecules, which are not expressed by resting or unactivated cells, can serve as an indicator or "footprint" of cellular activation. As will be described more fully herein infra, it has been discovered that IL-2R is produced in the soluble form and circulated in the body fluid as a result of immune-activation or under certain malignant conditions, particularly in lymphoreticular malignancies.
Heretofore, the measurement of IL-2 receptors (IL-2R) was limited to the assessment of cell-associated IL-2R by a variety of immunological techniques, involving several monoclonal antibodies against the human IL-2R. The observation of a soluble or released form of this molecule has heretofore not been reported. Therefore, this soluble molecule has never been measured in a body fluid. Hence, the detection of a soluble IL-2R molecule in humans under certain conditions is a new discovery.
With regard to cell-associated IL-2R, the quantitative measurement of IL-2R has been accomplished by binding of the monoclonal antibody followed by cellular analysis with a fluorescence-activated cell sorter (FACS) or similar machine (costing approximately $80,000-200,000). Such an analysis provides information about the number of cells expressing receptor and the amount of antibody bound, but does not provide a quantitative estimate of the number of receptors. The latter can be accomplished with an assay using radiolabeled monoclonal of known specific activity. However, such tests are tedious and require radionuclides which pose problems of safety and disposal.
In contrast, the Enzyme-Linked-Immunosorbent Assay (ELISA) of the present invention for IL-2R described in detail herein is quantitative, rapid, sensitive, inexpensive, and employs enzyme rather than radioactivity to detect IL-2R. The necessary reagents could be packed in a rather small box or kit and would have a "shelf-life" of about one year. The instrument necessary to "read" the assay can be a simple spectrophotometer which is available in virtually every clinical hospital laboratory. In addition to the method described herein, this solid-phase immunoassay has also been performed with alkaline phosphatase conjugated monoclonal antibody (7G7/B6) directly, instead of the FITC-conjugated 7G7/B6 and alkaline phosphatase conjugated rabbit anti-FITC.